SUMMARY/ABSTRACT Clinical development of novel CART cell therapies is hampered by low yield and poor functionality of peripheral blood T cells from many elderly and heavily-pretreated cancer patients. A proposed solution is the procurement of normal donor PBMC to manufacture a ?universal? T cell product that can be engineered to optimize persistence; however, certain histocompatibility barriers pose a unique challenge for successful engraftment. First generation universal CART (UCART) cell therapies utilized gene editing technology to disrupt the T cell receptor (TCR) in order to avoid graft-versus-host disease in recipient patients, but the allogeneic cells demonstrated limited persistence in the treatment of two infants with refractory ALL, most likely due to recognition of MHC II by host CD4+ T cells or missing-self response by host NK cells. One critical correlate of sustained remissions in patients with leukemia is the persistence of the CART cells, therefore next generation UCART cells must employ gene editing strategies to evade rejection by host CD8+ and CD4+ T cells as well as NK cells. In order to improve the persistence of allogeneic T cells, we will generate triple edited human and canine T cells through CRISPR/Cas9-mediated disruption of B2M, CD74, and TRAC to generate MHCInull, MHCIInull, TCRnull T cells and investigate the effect of these edits on allogeneic rejection in mixed lymphocyte cultures. We anticipate that MHCnull T cells will be rejected by stimulating the missing-self response of NK cells and will design and identify optimal HLA-E-B2M-peptide single chain trimer (SCT), as well as DLA- 79-B2M- peptide SCT, constructs as decoy MHC molecules to protect allo-T cells from NK lysis. We will evaluate the persistence of triple edited allogeneic canine UCART cells in dogs with B cell NHL and deplete NK cells in order to enhance the persistence of the UCART cells. We will also evaluate ?best in class? allo canine UCART cells in dogs with B-NHL through disruption of PD-1 or ectopic expression of optimal SCT on donor T cells. In order to develop a clinically translatable strategy for human UCARTs, we will compare the safety, persistence, and efficacy of first generation human UCART (TCRnull) and next generation, best in class UCART (TCRnull, MHCInull, MHCIInull + SCT) in xenogeneic settings of immunocompetent C57Bl/6J mice and in immunocompetent canine patients with spontaneous B-NHL. Success in this proposal will be measured through enhanced persistence of universal T cell therapies and will be a significant advancement to improve therapeutic durability and manufacturing of cellular immunotherapies.